The current level of research activity involving gene therapy with either synthetic vectors (carriers) or engineered viruses is unprecedented. Cationic liposomes (CLs) have emerged worldwide as among the most prevalent synthetic vectors employed in human clinical trials, due to their near lack of immune response and low toxicity. CL-vectors are also able to carry large pieces of DNA (consisting of entire genes and regulatory regions) into cells, which is not feasible with engineered viral vectors due to the limited size of virus capsids. CL_nucleic acid (CL_NA) complexes are employed both as DNA carriers and as siRNA (short interfering RNA; for gene silencing) carriers. However, the transfection efficiency and silencing efficiency of CL-vectors compared to viral vectors remains low for in vivo applications. Improvement of the efficiencies of synthetic vectors intended for in vivo applications requires a knowledge of the structures of CL-DNA and CL-siRNA complexes (in particular, the electrostatic interactions stabilizing assemblies of membranes and double-stranded NAs) as well as a mechanistic understanding of their interactions with cell membranes and the events leading to release of DNA and siRNA inside the cell. The aims of this research application are (1) to develop a new class of multi-component surface-functionalized CL-NA complexes, which will enable a mechanistic understanding of the initial pathway of complex uptake by the cell and the subsequent release of the NA-containing complex into the cell interior (cytosol), and (2) to understand the influence of lipid shape and membrane elastic properties on the formation of a new class of CL-DNA complexes possessing the membrane shape desired for interactions with endosome membranes inside the cell that optimally facilitate endosomal escape. Modern methods of organic and peptide chemistry will be employed to synthesize distinct PEG-lipids for cell targeting and endosome escape properties. These will be strategically combined in order to prepare high efficiency PEGylated CL-DNA complexes. The structures of CL-NA complexes will be solved using modern synchrotron x-ray diffraction techniques at the Stanford Synchrotron Radiation Lightsource and cryo-electron microscopy at the National Resource for Automated Molecular Microscopy at the Scripps Research Institute. Live-cell imaging with state-of-the-art optical fluorescence microscopes will allow us to visualize the interactions between CL-NA complexes and cellular components. Their structures will be correlated to their biological activity by quantitative measurements of transfection efficiency and silencing efficiency. The broad, long-term objective of our research is to develop a fundamental science base (via mechanistic studies of interactions of CL-NA complexes and cells) that will lead to the design and synthesis of synthetic carriers of DNA and siRNA optimized for gene therapeutics and disease control.